The present invention is concerned with new 1-naphtholphthalein monophosphates and the salts thereof which, as enzyme substrates, form a coloured anion under the influence of esterases at alkaline pH values and especially under the influence of alkaline phosphatase (EC 3.1.3.1). The present invention is also concerned with processes for the preparation of these monophosphates and of the salts thereof, as well as with a process and agent for the detection and photometric determination of alkaline phosphatase.
Alkaline phosphatase (AP) occurs, for example, in the liver, bones, in the small intestine, in the kidneys, in the bile and, to a smaller extent, in the placenta. An increase of the AP activity in the plasma has a diagnostic importance especially for the detection of liver diseases and diseases of the bone skeleton. For example, increased AP levels are observed in the case of Paget's disease, osteosarcomas, jaundice, hepatitis and similar diseases.
Over and above their physiological place value, alkaline phosphatase has, in recent years, achieved importance as a diagnostic adjuvant. Thus, for example, this enzyme is used as an indicator enzyme for enzyme immunoassays.
Thus, clinical chemistry and diagnosis require a method for the quantitative determination of alkaline phosphatase which is simple to use and, at the same time, gives results with good reproducibility. As a rule, for this purpose, there is used the direct photometric or fluorometric determination of compounds which are obtained from added specific substrates by hydrolytic cleavage brought about by AP. The compounds liberated by alkaline phosphatase are chromogenic or mesomerism-stabilised alcohols, especially phenols, and phosphate residues. As a rule, the former are determined qualitatively or quantitatively.
Not only in the case of colorimetric determinations but also in the case of fluorometric determinations, a parallel or subsequent carrying out of a blank value (without sample, i.e. without AP) is advisable in order to be able to recognise interferences due to a possible absorption possibly of unreacted substrate.
Besides p-nitrophenyl phosphate, naphthyl phosphates and phenolphthalein diphosphoric acids and appropriate derivatives thereof, phenolphthalein mono phosphoric acid and the salts thereof can be used as AP substrates (see U.S. Pat. Nos. 3,331,857 and 3,331,862). However, these compounds are unsatisfactory for the determination of alkaline phosphatase especially since the detection of the liberated amount of phenolphthalein is falsified by other compounds present in the sample. Thus, numerous serum components, for example bilirubin, have, like the mentioned phenolates liberated from the AP substrates, an absorption maximum at about 400 to 450 nm.
Therefore, thymolphthalein monophosphoric acid and, because of their good water-solubility, the corresponding sodium and ammonium salts, are today preferably used as substrates for the determination of alkaline phosphatase (see Clin. Chem., 16, 431-436/1970; Clin. Chem., 19, 1135-1138/1973; Japanese Patent Specification No. 83-158,199). In the absorption or emission spectrum, liberated thymolphthalein displays a maximum absorption band at about 595 nm. However, it is a disadvantage in the case of these compounds that, on the one hand, they have an insufficient sensitivity in the case of intestinal and placental AP and, on the other hand, there must be carried out not a kinetic determination but rather an end point determination which includes a rebuffering step.
o-Cresolphthalein monophosphoric acid is a further phenolphthalein derivative which is today commonly used as an AP substrate (see U.S. Pat. No. 3,975,405). With the help of this substrate, there is possible not only a continuous but also a discontinuous determination of alkaline phosphatase since the liberated o-cresolphthalein, unlike thymolphthalein, absorbs in a pH range which is optimum for the AP determination. However, the spectroscopic determination of the liberated o-cresolphthalein is disturbed in the presence of haemolytic samples since the absorption maximum of the o-cresolphthalein anion lies at about 570 nm.
Thus, it is an object of the present invention to provide new substrates for the determination of alkaline phosphatases which, besides a sufficient water-solubility, possess the property of liberating hydrolysis products under the influence of the enzyme. The liberated products have absorption maxima which are as long-waved as possible and thus, in the case of photometric determinations, are not or only slightly influenced by other sample components such as, particularly, haemolysis products. Furthermore, the new AP substrats are to be sufficiently stable and obtainable in high purity by simple and economically interesting syntheses.
This object is achieved by the new 1-naphtholphthalein monophosphates according to the present invention which, under the influence of alkaline esterases and especially of alkaline phosphatases, are reacted to give anions absorbing in the long wavelengths.